The present invention relates to heterologous enzymes produced by recombinant strains of Bacillus licheniformis and, in particular, to a xylanase derived from Bacillus pumilus PRL B12 which is efficient for use in the biobleaching of lignocellulosic pulp, expression vectors and recombinant Bacillus licheniformis hosts for the expression of the xylanase, and the use of the xylanase in the biobleaching of lignocellulosic pulp.
A common objective in the manufacture of products (such as paper products) from lignocellulosic pulps is to provide a pulp from which the product produced has as high a final brightness as is possible. A major factor that limits such final brightness is the quantity of lignin present in the pulp.
Lignin in such lignocellulosic pulps is mostly bound up with hemicellulose in a lignocellulosic complex. A major interface between the lignin and the remainder of the carbohydrates of the lignocellulosic complex is formed by xylan (1,4-xcex2-D-xylan) which is bonded thereto. To remove the lignin from the pulp, these bonds must first be broken.
Conventionally, lignocellulosic pulps, such as wood pulp, are delignified by being chemically-cooked. While being extremely useful for its purposes, (up to ninety-five per cent of the lignin present may be removed therefrom by such chemical-cooking), chemical-cooking cannot, by itself, reach higher levels of delignification without severly attacking the carbohydrates in the pulp. Thus, cooking must be stopped before the loss of carbohydrates becomes too important and before further delignification has occurred. This remaining lignin imparts a brown color to the cellulosic fibers in the pulp, thereby preventing the product fabricated therefrom from having as high a final brightness as possible.
To obtain further delignification of the pulp after chemical-cooking, various xe2x80x9cbleachingxe2x80x9d sequences are used. Conventionally, such bleaching sequences include xe2x80x9cClassicalxe2x80x9d (or conventional) bleaching sequences and Elemental Chlorine Free bleaching sequences. These bleaching sequences include a delignification stage followed by a series of bleaching stages. In the delignification stage, the pulp is first subjected to a chlorine and/or chlorine dioxide treatment step, usually followed by an alkaline extraction step.
While being effective for facilitating the removal (xe2x80x9cdelignificationxe2x80x9d) of substantial quantities of lignin from the chemically-cooked pulp, treatment of the pulp nonetheless suffers from drawbacks. Perhaps the most troublesome of these drawbacks (especially in the case of classical sequences) is that their use results in discharges of chlorinated compounds which have been linked to the formation of absorbable organic halides (AOX). AOX compounds have been associated with environmental toxicity. It is believed that the. AOX concentration in effluents is directly linked to the quantity of chlorine and chlorine dioxide compounds that are used in the process. Thus, AOX levels have become commonly used as a standard in the industry for determining the content of chlorinated organics in the bleaching plant""s effluents. As such, industries, such as the wood pulp and paper industry, which make products from such pulp have come under increasing pressure and regulation to reduce both AOX production and the consumption of both chlorine and chlorine dioxide used during bleaching.
To reduce or eliminate AOX production and the consumption of Cl2 and ClO2 while still achieving acceptable levels of deligninification, it has been proposed to utilize various hemicellulolytic enzymes to facilitate lignocellulosic pulp delignification (in a process commonly referred to as xe2x80x9cbiobleachingxe2x80x9d). In particular, it has been proposed to use of a wide variety of diverse xylanases which are secreted by a range of various fungi and bacteria, including bacteria of the genus Bacillus, for treating the pulp. However, these attempts have had varying degrees of success depending upon the precise characteristics of the xylanase which has been employed therefor. To be successfully employed in commercial biobleaching applications, a xylanase should be efficient over the pH range naturally possessed by the pulp when the xylanase is utilized during biobleaching. Furthermore, the xylanase should be devoid of any residual cellulase activity.
In biobleaching, xylanase may be added to the pulp after it has exited the chemical-cooker, but before it has been chemically-treated. At that point, the pulp typically has a pH in the range of about 7.0 to 9.5. The identification and utilization of a xylanase which is efficient over this alkaline pH range would greatly reduce the control that must be maintained over that aspect of the process conditions as well as reduce the quantity of chlorine and reactive oxidants needed. to chemically-treat the chemically-cooked pulp.
It has been known for some years that microorganisms of the species Bacillus pumilus extracellularly secrete xylanases. Indeed, as early as 1960 it was disclosed that the culture media of B. pumilus contains xylanases which make this culture broth useful for food processing applications (see Canadian Letters Patent No. 603,953 and U.S. Pat. No. 2,821,501). However, nowhere do those patents either disclose, teach or suggest the isolation and/or purification of the enzymes (including the xylanases) from the culture broth into which they are secreted. Furthermore, it was reported therein that in pH""s above 8, those xylanases will generally be inactivated.
To the best of our knowledge, the xylanase of only two strains of Bacillus pumilus have ever been isolated and/or purified : Bacillus pumilus IPO and Bacillus pumilus DSM 6124. However, there is nothing to indicate whether the xylanase from B. pumilus PRL B12 would have potential usefulness in biobleaching applications.
Only one xylanase from any strain of B. pumilus has ever been proposed for use in biobleaching, and that xylanase was from a specially designed mutantxe2x80x94B. pumilus DSM 6124. The isolation and purification of the xylanase secreted from B. pumilus DSM 6124, as well as its use in biobleaching has been disclosed in International Publication No. WO 91/02839 and International Publication No. WO 92/03540. However, as reported therein, the xylanase of strain DSM 6124 has an optimum pH of only 5-7 and appears to be of limited efficiency for the delignification of pulps having pH""s of up to only about 8.5.
The presence, in the culture broth, of extracellular xylanases secreted by Bacillus pumilus PRL B12 and B. pumilus PRL B92 has also long been known, being reported as early as 1954 (1). It was further reported therein that, when in the milieu of the culture broth, the xylanase of B. pumilus PRL B12 is stable up to pH 11. However, nowhere does that reference either disclose, teach or suggest the isolation and/or purification of the xylanase from the culture broth into which it is secreted, nor is there any indication whatsoever in (1) as to what physical characteristics such an isolated and/or purified xylanase would have when not in the milieu of the culture broth. Furthermore, those xylanases have never been proposed for use in the biobleaching of chemical pulp.
It is known that, when in the culture broth into which they are secreted, xylanases are often contaminated by other enzymes. This can make the isolation and purification of the xylanase difficult and costly. This is particularly significant in that there is no information in (1) as to whether or not those xylanases ever were, or ever could be, isolated and/or purified, nor is there any information in (1) as to how difficult or successful one could expect such a task to be.
Furthermore, it is known that, in the culture broth, the contamination of the xylanase by other enzymes can effect the apparent physical characteristics of the xylanase, including its efficiency over different pH ranges. This aspect is particularly notable in light of the fact that xylanases exhibit a wide variety of characteristics, even when secreted from extensively homologous microorganisms of the same genus.
Thus, it can be seen that there still remains a need to locate and provide an isolated and/or purified xylanase which is efficient for the delignification of lignocellulosic pulp having a pH in the alkaline range of 7-9.5, so as to be useful for the pretreatment of lignocellulosic pulp by facilitating delignification of the lignocellulosic pulp before the bleaching thereof. In this fashion, the xylanase of the present invention permits a reduction in the quantity of chlorine, chlorine dioxide and other/or reactive oxidants which are needed to be utilized in the bleaching sequences for chemically-treating the lignocellulosic pulp, as well as a reduction in the production of AOX compounds, while still providing a lignocellulosic pulp that has an acceptably-low lignin content (as measured by the Kappa Index of the pulp).
It is further desireable to provide a host for expressing various enzymes, such as the xylanase, in good yields. In this regard, it is noted that if the enzyme yield from the host organism is too low, then enzyme production will be inefficient and too expensive.
While strains of B. pumilus are often cited as being hyper-producers of xylanase, the yields obtained therefrom are nonetheless too low to permit their use in industrial applications.
In order to obtain an increase in the yield of B. pumilus xylanase, it has been proposed to clone the xylanase gene(s) from Bacillus pumilus IPO into compatible hosts for achieving the heterologous expression of the xylanase in increased yields from the transformed hosts. The expression of the xylan-degrading genes of Bacillus pumilus IPO in species of Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae have all been disclosed. However, we are not aware of any disclosure of the usefulness of those transformed hosts for industrial applications.
In spite of the above references, it nonetheless still remains desireable to identify and prepare suitable hosts which are stable, capable of use in commercial applications and which are capable of extracellularly secreting xylanase, and in particular xylanase derived from B. pumilus PRL B12, in suitable yields.
Strains of Bacillus licheniformis are routinely used as hosts for the heterologous extracellular expression of various neutral and acidic enzymes (mainly proteases and alpha-amylases) in large scale industrial situations.
While success has been achieved in obtaining the heterologous expression of these neutral and acidic enzymes noted above, the use of the strains of B. licheniformis for the heterologous production of enzymes, such as xylanase, under alkaline conditions nonetheless presents several drawbacks. One of these drawbacks is that B. licheniformis is a producer of alkaline proteases, which can degrade xylanases and other enzymes under alkaline conditions. Such a feature is an obvious disadvantage and disincentive to the use of B. licheniformis as a host for the secretion of a xylanase. Further, even if making the deletions of the alkaline protease gene had been suggested, there would be no guarantee of success, especially in light of International Publication No. WO 91/02792, wherein it was reported that the presence of even a part of a protease gene can induce a reduction in the productivity of heterologous enzymes by the strain. Accordingly, to the best of our knowledge, no one has suggested the use of strains of Bacillus licheniformis for the heterologous expression of xylanase.
Thus, it can be seen that there further remains a need to provide a stable host capable of expressing such a xylanase in high yields.
It is a primary object of the present invention to identify, purify and provide a xylanase which is efficient for facilitating the delignification of lignocellulosic pulp, and in particular wood pulp, having a broad alkaline pH range and, in particular, a preferred pH range of 7.0-9.5.
It is another primary object of the present invention to identify and prepare suitable hosts which are stable, easy to use in commercial applications and which are capable of extracellularly secreting, in suitable yields, various enzymes, and in particular xylanase, especially xylanase derived from B. pumilus PRL B12, under alkaline conditions.
It is a further object of the present invention to isolate and purify the gene (nucleotide sequence) that codes for the xylanase of the present invention, as well as to provide an appropriate vector therefor which is useful when transformed in a suitable host, such that heterologous expression of the xylanase of the present invention may be achieved.
A still further object of the present invention is to provide expression vectors which include the nucleotide that codes for the xylanase of Bacillus pumilus PRL B12 and methods for the preparation thereof.
In still another aspect of. the present invention, a further object is to provide expression hosts for the expression vectors of the xylanase of the present invention, as well as for other enzymes, such as alpha-amylase, pullulanase, subtilisin and alkaline protease.
In still yet another aspect of the present invention, a further object is to provide an enzymatic treatment employing such a xylanase which permits a reduction in the quantity of chlorine, chlorine dioxide and other reactive oxidants used for delignification during biobleaching, as well as a reduction in the production of AOX compounds while still permitting a pulp to be obtained that has an acceptably-low lignin content.
In accordance with the teachings of the present invention, a purified xylanase that is derived from B. pumilus PRL B12 and mutants and variants thereof, is disclosed. This xylanase consists essentially of the amino acid sequence of amino acids numbered 1 to 200 of FIGS. 1a and 1b and mutants and variants of this amino acid sequence. This xylanase is efficient for facilitating the delignification of pulp having a pH, preferably, in the range of.7.0 to 9.5.
Alternatively, a purified xylanase is disclosed having a molecular weight of about 26 kDa as determined by an SDS-PAGE gel electrophoresis method as defined herein, an isoeletric point of about 9.8-9.9 in terms of a value as measured by an isoeletric focusing method as defined herein, an optimum temperature of about 55xc2x0 C. as measured by a xylan hydrolysis assay method as defined herein and an optimum pH of about 6.5-7.5 as measured by a xylan hydrolysis assay method, as defined herein.
In another alternative, a purified xylanase is disclosed that has a molecular weight of about 26 kDa as determined by an SDS-PAGE gel electrophoresis method as defined herein and a molecular weight of about 22,500 kilodaltons (and more precisely about 22,534 kilodaltons) as deduced from the amino acid sequence of the mature xylanase by a deduction method as defined herein.
Finally, it is noted that the purified xylanase disclosed herein has, for the biobleaching of lignocellulosic pulp, an optimum xylanolytic activity of about 7.5-8.5 as measured by the kappa index of the lignocellulosic pulp treated thereby in a method defined herein.
By the term xe2x80x9cas defined hereinxe2x80x9d what is meant is the method defined in the various examples set forth below.
By the term xe2x80x9cderived fromxe2x80x9d when used in reference to B. pumilus PRL B12 and the xylanase and the nucleotide sequences disclosed herein, what is meant are the xylanase and the nucleotide sequences which are native to (or which are identical to those xylanase and nucleotide sequences which are native to) B. pumilus PRL B12.
In further accordance with the teachings of the present invention, a xylanase is disclosed herein that is heterologously-produced by and obtained from a microorganism of the genus Bacillus of the type which has a naturally-occurring alkaline protease gene. It is preferred that such hosts be aerobic. It is further preferred that such hosts not be thermophilic. Examples of such hosts include microorganisms of the species Bacillus subtilis and Bacillus licheniformis. Other examples include microorganisms of the species Bacillus alkalophilus, Bacillus lentus and Bacillus amyloliquefaciens. It is further preferred that this xylanase be heterologously-produced by and obtained from such a microorganism which has had the alkaline protease gene deleted therefrom, such as B. licheniformis SE2 delap1, B. licheniformis SE2 delap3, B. licheniformis SE2 delap6 and B. subtilis SE3. It is still further preferred that the xylanase be expressed heterologously by, and obtained from, strains of such microorganisms which have been transformed to include the xylanase coding sequence (and preferably, the entire xylanase gene) of B. pumilus PRL B12, so that the xylanase can be expressed thereby.
Alternatively, disclosed herein is a xylanase obtained from a strain of Bacillus pumilus, such as B. pumilus PRL B12.
In still further accordance with the teachings of the present invention, a process is disclosed herein for producing a xylanase in a Bacillus species. The process includes transforming a suitable strain of a Bacillus with a nucleotide sequence coding for at least the mature portion of the xylanase (SEQ ID NO:34). In this manner, a Bacillus having a complete xylanase gene expression unit is formed. The Bacillus is then cultured under suitable conditions for the expression of xylanase. Finally, the process disclosed involves recovering the xylanase from the culture. In a preferred embodiment, the Bacillus is Bacillus licheniformis. It is further preferred that the nucleotide sequence is derived from Bacillus pumilus PRL B12.
In yet further accordance with the teachings of the present invention, the gene (DNA molecule) coding for the xylanase of B. pumilus PRL B12 has been isolated therefrom and purified. In this regard, the nucleotide sequence of the entire xylanase gene of B. pumilus PRL B12 is disclosed. This nucleotide sequence includes those nucleotides coding for the mature xylanase, as well as those nucleotides coding for the precursor xylanase (SEQ ID NO:31) of B. pumilus PRL B12 (SEQ ID NO:32). This nucleotide sequence further includes promoters of the gene, as well as upstream and downstream nucleotide sequences.
In still further accordance with the teachings of the present invention, suitable expression vectors are disclosed herein. A preferred expression vector having, as a component thereof, the nucleotide sequence coding for the mature xylanase is disclosed, in addition to methods for the preparation thereof. Alternatively, this expression vector can further include the nucleotide sequences (SEQ ID NO:30) coding for the precursor xylanase. If desired, the upstream and/or downstream coding sequences of the xylanase gene may also be included. Most preferably, this expression vector includes the entire xylanase gene (SEQ ID NO:35) seen in FIGS. 1a and 1b. This most preferred expression vector is pUB-BPX12.
Other expression vectors disclosed herein include pUBDEBRA1, pKAC1, pLI1, pL7SBT, pL7TAKA, each of which code for various other proteins, so as to permit the heterologous expression thereof by the expression hosts disclosed herein.
In still further accordance with the teachings of the present invention, suitable hosts have been identified and methods for the preparation thereof have been disclosed. The hosts are stable, are able to operate effectively under varying process conditions and are capable of the heterologous production of enzymes, including xylanase derived from B. pumilus PRL B12, in good yields. Preferably, these hosts include (biologically pure cultures of) strains of Bacillus licheniformis and, in particular, the strain designated herein as B. licheniformis SE2 delap1 and mutants and variants thereof, such as the strains designated herein as B. licheniformis SE2 delap3 and mutants and variants thereof and B. licheniformis SE2 delap6 and mutants and variants thereof. Disclosed herein are also methods for preparing such strains of B. licheniformis by performing chromosomal deletions of the gene(s) thereof which code for alkaline protease. In this regard, deletion plasmids LD1, LD3 and LD6, and the methods for the construction thereof, are also disclosed herein.
In yet further accordance with the teachings of the present invention, the use of the xylanase of the present invention is disclosed herein as a treatment in the biobleaching of lignocellulosic pulp, such as Kraft wood pulp. Preferably, the xylanase may be used as a pretreatment in conjunction with traditional bleaching sequences. More particularly, use of the xylanase is disclosed as a pretreatment to classical bleaching sequences of the type CEDPD and C/DEDPD, as those sequences are defined herein. Also more particularly, use of the xylanase is disclosed as a pretreatment in conjunction with Elemental Chlorine Free Sequences of the type DEDPD, as those sequences are defined herein.
In still yet further accordance with the teachings of the present intention, the use of the xylanase of the present invention is disclosed herein in the biobleaching of lignocellulosic pulp in Totally Chlorine Free sequences. More particularly, use of the xylanase in Totally Chlorine Free sequences of the type OQPZP. It is especially preferred that, in such sequences, the xylanase be used in conjunction with the sequestrant (Q)step to give the sequence OX/QPZP.
It is especially preferred to use this xylanase in the sequences described above for the biobleaching of wood pulp and, more particularly, kraft wood pulp.
In still yet further accordance with the teachings of the present invention, an enzymatic treatment employing the xylanase is disclosed. Preferably, such a treatment is a pretreatment for classical and Elemental Chlorine Free bleaching sequences or in conjunction with one of the steps of a Totally Chlorine Free bleaching sequence. This enzymatic treatment permits a reduction in the quantity of chlorine, chlorine dioxide and/or other reactive oxidants needed to be used for subsequently chemically-treating the lignocellulosic pulp, as well as a reduction in the production of AOX compounds, while still permitting a pulp to be obtained which has an acceptably-low lignin content. Furthermore, this enzymatic pretreatment either: (a) does not diminish the final product brightness achieved (in the case of classic sequences or ECF sequences); or (b) still provides a final product brightness which is satisfactory while employing acceptable quantities of reactants (in the case of TCF sequences).
The use of the xylanase of the present invention in the treatment (biobleaching) of lignocellulosic pulp is usually done in a separate stage. It may also be done in combination with sequestrants. The xylanase can therefore be used not only to reduce the chemical charges of chlorine, chlorine dioxide and/or other reactive oxidants, such as oxygen, ozone or peroxide, but it also decreases the costs of the biobleaching process.